The present invention concerns a recombinant adenoviral vector derived from an adenovirus genome in which at least a part of the E3 region is deleted or is non functional, wherein said adenoviral vector retains E3 sequences encoding a functional 14.7K protein, a functional 14.5K protein, and/or a functional 10.4K protein. The present invention further relates to the use of a polynucleotide comprising at least one or more gene(s) of an E3 region of an adenovirus, taken individually or in combination, to protect from an inflammatory reaction in a host cell, tissue or organism. The present invention additionally concerns a viral particle, a host cell and a composition comprising said recombinant adenoviral vector or said polynucleotide, as well as their use for therapeutic or prophylactic purpose. The invention is of very special interest in gene therapy applications and in the protection from TNF (Tumor necrosis factor) or Fas-mediated inflammatory conditions.
Gene therapy can be defined as the transfer of genetic material into a cell or an organism. The possibility of treating human disorders by gene therapy has changed in the last few years from the stage of theoretical considerations to that of clinical applications. The first protocol applied to man was initiated in the USA in September 1990 on a patient suffering from adenine deaminase (ADA) deficiency. This first encouraging experiment has been followed by numerous new applications and promising clinical trials based on gene therapy are currently ongoing. The large majority of the current protocols employ vectors to carry the therapeutic gene to the cells to be treated.
There are two main types of gene-delivery vectors, viral and non-viral. Viral vectors are derived from naturally-occuring viruses and use the diverse and highly sophisticated mechanisms that wild-type viruses have developed to cross the cellular membrane, escape from lysosomal degradation and deliver their genome to the nucleus. Many different viruses are being adapted as vectors, but the most advanced are retrovirus, adenovirus and adeno-associated virus (AAV) (Robbins et al., 1998, Trends Biotechnol. 16, 35-40; Miller, 1997, Human Gene Therapy 8, 803-815; Montain et al., 2000, Tibtech 18, 119-128). Substantial effort has also gone into developing poxviruses (especially vaccinia) and herpes simplex virus (HPV). Non-viral approaches include naked DNA (i.e., plasmidic DNA; Wolff et al., 1990, Science 247, 1465-1468), DNA complexed with cationic lipids (for a review, see for example Rolland, 1998, Critical reviews in Therapeutic Drug Carrier Systems 15, 143-198) and particles comprising DNA condensed with cationic polymers (Wagner et al., 1990, Proc. Natl. Acad. Sci. USA 87, 3410-3414 and Gottschalk et al., 1996, Gene Ther. 3, 448-457). At the present stage of development, the viral vectors generally give the most efficient transfection but their main disadvantages include their limited cloning capacity, their tendency to elicit immune and inflammatory responses and their manufacturing difficulties. Non-viral vectors achieve less efficient transfection but have no insert-size limitation, are less immunogenic and easier to manufacture.
Adenoviruses have been detected in many animal species, are non-integrative and of low pathogenicity. They are able to infect a variety of cell types, dividing as well as quiescent cells. They have a natural tropism for airway epithelia. In addition, they have been used as live enteric vaccines for many years with an excellent safety profile. Finally, they can be easily grown and purified in large quantities. These features have made adenoviruses particularly appropriate for use as gene therapy vectors for therapeutic and vaccine purposes. Their genome consists of a linear double-standed DNA molecule of approximately 36 kb carrying more than about thirty genes necessary to complete the viral cycle. The early genes are divided into 4 regions dispersed in the adenoviral genome (E1 to E4). The E1, E2 and E4 regions are essential for viral replication. Early region 3 (E3) has been termed a xe2x80x9cnon essential regionxe2x80x9d based on the observation that naturally occuring mutants or hybrid viruses deleted within the E3 region still replicate like wild-type viruses in cultured cells (Kelly and Lewis, 1973, J. Virol. 12, 643-652). The late genes (L1 to L5) encode in their majority the structural proteins constituting the viral capsid. They overlap at least in part with the early transcription units and are transcribed from a unique promoter (MLP for Major Late Promoter). In addition, the adenoviral genome carries at both extremities cis-acting regions essential for DNA replication, respectively the 5xe2x80x2 and 3xe2x80x2 ITRs (Inverted Terminal Repeats) and a packaging sequence.
The E3 region spans map units (MU) 76.6-86.2 (nucleotides 27329 to 31103 in Ad5) and is controlled by its own promoter (E3 promoter) that is quite stringently dependent on the presence of E1 transcription factors for expression. Transcription occurs from left to right with regards to the adenoviral genome (sense orientation) and produces a variety of different mRNA species which differ both in their splicing patterns and in poly A site utilization. Among the nine proteins which are potentially encoded by the mRNAs which initiate from the E3 promoter, seven have been clearly identified. They have been named according to their estimated molecular weight, respectively 19, 14.7, 14.5, 12.5, 11.6, 10.4 and 6.7 kDa. To date, the function of only five of them can be assigned. The E3 11.6K protein is involved in the lysis of adenovirus-infected cells (Tollefson et al., 1996, J. Virol. 70, 2296-2306) whereas the E3-gp19K, 10.4K, 14.5K and 14.7K proteins are immunomodulatory proteins allowing an attenuation of the host immune response against adenovirus-infected cells.
The best characterized of the E3 protein, E3-gp19K, is an integral membrane protein anchored in the membrane of the endoplasmic reticulum (ER). In vitro studies have established that the E3 gp19K protein blocks cytolysis by CTLs (Cytotoxic T lymphocytes) by binding major histocompatibility complex (MHC) class I antigens (Signas et al., 1982, Nature 299, 175-178). This interaction results in the retention of class I molecules in the ER, thus preventing their cell-surface expression (Burgert et al., 1985, Cell 41, 987-997) and, ultimately, recognition of adenovirus-infected cells by CTLs (Andersson et al., 1987, J. Immunol. 138, 3960-3966).
Tumor Necrosis Factor xcex1 (TNFxcex1) has been shown to be important for adenovirus clearance during infection. TNFxcex1 is a potent cytokine responsible for a wide variety of physiologic and immunologic effects. It is secreted by activated macrophages and lymphocytes in response to virus infections, tissue damages, bacterial endotoxins and other cytokines. TNFxcex1 binds to specific receptors, leading to activation of signal transduction pathways, transcription factors and protein kinases. In addition, TNFxcex1 is cytotoxic to a wide variety of primary tumors and transformed cell lines (Browning and Ribolini, 1989, J. Immunol. 143, 1859-1967). TNFxcex1 can also suppress the replication of both DNA and RNA viruses in infected cells (Mestan et al., 1986, Nature 323, 816-819). TNF activates phospholipase A2 (PLA2), resulting in the release of arachidonic acid (AA) which are responsible for the establishment of an inflammmatory status.
A number of experimental evidences suggest that three E3-encoded proteins, respectively 14.7K, 10.4K and 14.5K inhibit TNFxcex1-induced cytolysis and TNF-induced release of AA (Krajcsi et al., 1996, J. Virol. 70, 4904-4913). The 14.7K protein is a hydrophilic protein found in the soluble fractions of both cytosol and nucleus of adenovirus-infected cells. The mechanism by which the 14.7K protein inhibits TNFxcex1-mediated cytolysis, is not fully defined but it probably interferes with the TNFxcex1 receptor signaling pathway.
E3 10.4K and E3 14.5K proteins are integral membrane proteins that act as a complex (named RID complex for receptor internalization and degradation) to protect cells from the lytic effect of TNFxcex1 (Gooding et al., 1991, J. Virol. 65, 4114-4123). These proteins have an additional function in cell surface receptor modulation and have been shown to accelerate internalization of the epidermal growth factor receptor, the insulin receptor and Fas receptor (Fas) by targeting them to lysosomes for degradation (Steward et al., 1995, J. Virol. 69, 172-181; Shisler et al., 1997, J Virol. 71, 8299-8306; Tollefson et al., 1998, Nature 392, 726-730). The nature of the interaction between the RID complex and these cellular proteins is unknown. Fas is expressed on numerous tissues and especially on T cells, hepatocytes, heart and kidney cells. It is central in the homeostasis of a number of organs as well as the immune system but also in the elimination of virus-infected cells by CTLs.
The redundant anti-TNF functions encoded by the adenoviruses leads are presumed to be relevant to viral pathogenesis. The actual contribution of the E3-encoded TNFxcex1 antagonists to the maintenance of the virus in an infected host has not yet been investigated, apart from the observation that expression of the E3-14.7K gene in the respiratory epithelium of transgenic mice reduces lung inflammation and enhances adenoviral vector gene expression.
Moreover, TNFxcex1 and Fas are also implicated in a number of pathological conditions. For example, high levels of TNFxcex1 are associated with acute hepatotoxicity in many animal models including lipopolysaccharides (LPS) and ConA-induced liver injury. It is an important mediator in septic shock and fulminant hepatic failure (Jo et al., 2000, Nat. Med. 6, 564). High levels of circulating TNF have poor prognostic values in patients with viral hepatitis B and C or with alcoholic liver disease (reviewed in Bradham et al., 1998, The American Journal of Physiology 275, 4387). Cumulative evidence suggests the contribution of Fas associated with Fas ligand (FasL) to inflammatory and tissue-damages. A role of these molecules has been shown in alcohol-induced cirrhosis, hepatitis, graft rejection and autoimmune diseases.
The adenoviral vectors presently used in gene therapy protocols lack most of the E1 region which renders the viruses replication-deficient to avoid their dissemination in the environment and the host organism. Moreover, most of the adenoviral vectors are also E3 deleted, in order to increase their cloning capacity. The feasability of gene transfer using these vectors has been demonstrated into a variety of tissues in vivo (see for example Yei et al.,1994, Hum. Gene Ther. 5, 731-744; Dai et al., 1995, Proc. Natl. Acad. Sci. USA 92, 1401-1405; Howell et al., 1998, Hum. Gene Ther. 9, 629-634; Nielsen et al., 1998, Hum. Gene Ther. 9, 681-694; U.S. Pat. Nos. 6,099,831; 6,013,638). However, their use is associated with acute inflammation and toxicity in a number of animal models (Yang et al., 1994, Proc. Natl. Acad. Sci. USA 91, 4407-4411; Zsengeller et al., 1995, Hum. Gene Ther. 6, 457-467) as well as with host immune responses to the viral vector and gene products (Yang et al., 1995, J. Virol. 69, 2004-2015), resulting in the elimination of the infected cells and transient gene expression.
The persistence of gene expression is a prerequisite before envisaging the wide use of adenoviral vectors in human gene therapy protocols, in particular in view of treatment of chronic and genetic diseases. With the goal of improving adenovirus-mediated gene expression, it has been suggested to engineer adenoviral vectors expressing the E3-encoded proteins and the presently available studies have been conducted with the entire E3 region. The European patent application EP707071 discloses recombinant adenoviruses having a foreign gene inserted in replacement of the E1 region and retaining the full-sized wild-type E3 region driven by its own promoter. Experimental data demonstrate the capability of these E1xe2x88x92 E3+ adenoviruses to express the foreign gene product and provide therapeutic effect in various animal models, but, in the absence of any comparative data, the benefit of retaining the entire E3 region over an E3-deleted virus was not clearly established. Long-term gene expression and attenuation of the antiviral immune response was observed in a rat model injected with a E1-deleted recombinant adenovirus containing the entire E3 region driven by a strong and constitutive promoter (Ilan et al., 1997, Proc. Natl. Acad. Sci. USA 94, 2587-2592). However, none of these studies have investigated whether the maintenance of the entire E3 region has a protective effect on the inflammation and toxicity generally observed with the conventional adenoviral vectors which are responsible for rapid elimination of the infected host cells, a transient gene expression and activation of pro-inflammatory substances that have pleiotropic effects.
The invention provides adenoviral vectors for gene therapy that retain the gene(s) of the E3 region encoding the 14.7K protein and/or the RID complex (formed by the association of the 10.4K and 14.5K proteins). It was surprisingly found that, when used in a murine model of TNF-induced liver pathology, the 14.7K-expressing adenoviral vector protects the animal from death by inflammatory reactions. In a similar model, the RID-expressing adenoviral vector inhibits acute hepatitis induced by an anti-Fas antibody. These results validate the functionality of these vectors for protecting infected cells, tissues or organisms from inflammation.
Thus, the technical problem underlying the present invention is the provision of recombinant adenoviral vectors which do not have a number of drawbacks associated with conventional vectors disclosed for this purpose so far and of means which allow protection from an inflammation condition, especially mediated by TNF or Fas or induced by administration of gene therapy (adenoviral) vectors.
This problem is solved by the provision of the embodiments characterized in the claims.
Accordingly, the present invention relates to a recombinant adenoviral vector derived from an adenovirus genome in which at least a part of the E3 region is deleted or is non functional, wherein said adenoviral vector retains E3 sequences encoding:
(i) a functional 14.7K protein,
(ii) a functional 14.5K protein,
(iii) a functional 10.4K protein, and/or
wherein said recombinant adenoviral vector comprises a gene of interest, and wherein said retained E3 sequences and said gene of interest are operably linked to regulatory elements allowing their expression in a host cell.